Various scientific and scholarly articles are referred to in parentheses throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains. Full citations of the references appear at the end of the specification.
Eggplant (Solanum melongena L.) is an important crop species in Europe, Asia and North America. However, in some parts of the world its commercial production is frequently hampered by devastating attacks by the Colorado potato beetle (CPB, Leptinotarsa decemlineata [Say]). In the absence of an effective pest control program, the CPB may cause total destruction of eggplant crops. Chemical pesticide application is costly to farmers and damaging to the environment.
Attempts have been made to produce eggplant resistant to the CPB. For instance, the native cryIIIB Bacillus thuringiensis gene was used for transformation of eggplant (Chen et al., 1995). The native cryIIIB gene encodes a protein in spores of Bacillus thuringiensis (Bt) which is toxic to certain coleopteran insects, including CPB. A transfer of this cryIIIB gene via the recombinant DNA approach was expected to produce sufficient amounts of toxic protein in eggplants to provide an adequate level of resistance to CPB. However, field tests of more than 200 primary transgenic eggplants and their R.sub.1 progeny containing the cryIIIB gene failed to demonstrate any noticeable resistance to the CPB. It appears that this gene was poorly expressed when incorporated into eggplant. The experimental evidence suggests that the transcription process of the native Bt genes in certain plant cells generates fragmented mRNA, instead of entire transcripts, which is non-functional in the translation process (Murray et al., 1991; Van Aarssen et al., 1995). In these instances the lack of CPB resistance in transgenic plants is directly related to the absence of toxic protein synthesis in the cells.
Analysis of the coding sequences of a number of native Bt genes has revealed structural features not found in most plant genes (Perlak et al., 1991; Sutton et al., 1992; Adang et al., 1993). For instance, along the coding region of the native Bt gene, there are frequent stretches of multiple A's and T's. In addition, the GC content of the native Bt gene is much lower than in plant genes. The polyadenylation signal sequence AATAAA of plant genes and the mRNA destabilizing ATTTA sequence is often found in the middle of the coding region. Also, the codon usage of the native Bt gene is different from many plant genes.
Modification of sequences in the native Bt genes by nucleotide substitution has been undertaken with the goal of making the Bt coding sequence more similar to plant gene coding sequences. Perlak modified the lepidopteran cryIA(b) and cryIA(c) Bt genes by changing 3% and 20%, respectively, of the nucleotides (Perlak et al., 1990; Perlak et al., 1991). In comparison to the corresponding native genes, the toxic protein production from the synthetic Bt genes in transgenic cotton and tomato was increased. Modified versions of the coleopteran cryIIIA gene have been engineered by Sutton et al. (1992) and Adang et al. (1993) who changed the coding sequence by 17% and 11% respectively. The modified genes enhanced production of the toxic protein in tobacco (Sutton et al., 1992) and potato cells (Adang et al., 1993), enhancing their resistance to a representative insect pest.
Recently, it has been reported that modified cryIA(b) and cryIC genes were highly expressed in tomato (van der Salm et al., 1994), syn cryIA in corn (Armstrong et al., 1995), and syn cryIA(b) in rice (Wunn et al., 1996). These results convincingly demonstrated that recovery of a high level of pest resistance in plants could be achieved by using modified Bt genes rather than native ones.
Transfer of genetic information into the genome of a plant species by recombinant DNA techniques has become an important strategy in basic studies of plant biology as well as in the improvement of cultivated plants. However, a severe impediment to the applicability of this approach with many plant species is the inefficacy of a reliable transformation and regeneration procedure in vitro. A review (Van Wordragen and Dons, 1992) describes Agrobacterium tumefaciens-mediated transformation of plant species, still the most widely used transfer system in plants, and indicates that only a few species (model plant species) can be transformed and regenerated routinely with commonly used procedures. The great majority of plants, the so-called recalcitrant species, require empirical development of a particular transformation protocol for a particular species. In developing such species-specific transformation protocols, efficiency is an important attribute in evaluating various protocols. This is of particular importance in cases where it is necessary to secure a large population of transformed genotypes for subsequent screening and selection of desired traits.
Eggplant is a species that has been recalcitrant to transformation. Rotino and Gleddie (1990) reported a transformation efficiency of only seven percent. Neither those methods, nor the methods of Guri and Sink (1988) have been demonstrated to result in transformation efficiencies greater than 7%. Thus, improved methods are needed for high efficiency transformation of eggplant, especially with useful genes, such as cryIIIA and other genes suitable for expression in plants, for the purpose of generating transgenic varieties that are resistant to insects or other plant pathogens.